The preservation of eukaryotic cells, tissues or organs is commonly carried out by chilling them sufficiently to slow or halt metabolic processes that require ongoing support by an organism or the environment to maintain viability. The preservation of cells, tissues or organs by such chilling is generally referred to as hypothermic preservation or hypothermic storage.
There are two broad types of hypothermic storage for cells, tissues or organs. The first involves storage at temperatures above the freezing point of the solution or medium in which the sample is suspended, immersed or with which the sample is perfused. For example, such temperatures may be in the range of about 17° C. to 0° C. Such conditions are appropriate only for short term storage, generally on the order of hours to several days to about a week.
The second broad type, cryogenic storage, involves storage at lower temperatures (below 0° C.), for example, as low as −80° C. to −196° C. Storage under these conditions is more appropriate for longer periods of time. In this second broad category, there are two general subclasses of preservation approach. The first sub-class involves the freezing of the samples in a medium or solution that permits the formation of ice crystals. For this approach, cryoprotective agents (e.g., dimethyl sulfoxide (DMSO), glygerol) are added that mitigate the effect of the formation of ice crystals, essentially by causing dehydration of the cells that prevents intracellular water crystal formation. This approach will be referred to herein as “cryopreservation.”
The second sub-class involves immersing or suspending cells or tissues in, or perfusing organs with, a solution comprising an agent that prevents ice nucleation within the extracellular and intracellular environment thereby preventing ice formation Reducing the temperature of such a solution below its glass transition temperature (Tg) results in the formation of a glass or vitreous state, which is defined as an amorphous solid without crystalline structure. That is, the second sub-category involves preservation in a solution that forms a glass or vitreous state, instead of freezing in a crystalline state. In this approach, the cell, tissue or organ never comes in contact with or experiences the formation of extra- or intracellular ice crystals. This approach is referred to herein as “vitrification.” In such a solution, an amorphous glass forms when the solution is cooled below the glass transition temperature (Tg), and the formation of this amorphous glass or vitreous state precludes the subsequent alignment of the water molecules that is necessary for the formation of crystalline ice, even when temperatures are reduced below the homogeneous nucleation temperature.
The basic challenge of hypothermic storage is to preserve the material in a state that can be reversed without causing extensive cell damage or cell death. Approaches that minimize the formation of ice crystals in or around cells are well known to aid in the survival and ultimate recovery of material stored at hypothermic temperatures. However, even when ice crystal formation is essentially completely avoided, as in the vitrification approach, there is an associated degree of cell death. Cell death is known to occur by two different mechanisms. The first, necrotic cell death or necrosis, is not mediated by a specific cellular pathway. Necrosis is characterized by the loss of cell membrane integrity resulting in cell swelling, and is caused by a number of pathological agents. DNA in cells that undergo necrosis is cleaved in a random fashion. Thus, the DNA from cells that have undergone necrosis appears as a continuous smear when subjected to gel electrophoresis. The second cell death mechanism, apoptosis or programmed cell death, is the result of the activation of a specific biochemical pathway involving a cascade of biochemical activation steps that ultimately result in the death of the cell. Apoptosis is characterized by cell shrinkage, intact plasma membranes, and non-random cleavage of DNA at an approximately 180 nucleotide interval, evidenced by a ladder of DNA cleavage products upon gel electrophoresis of genomic DNA. Apoptosis is reviewed, for example, by Kerr et al., 1994, Cancer 73: 2013 and Evan & Littlewood, 1998, Science 281: 1317.
It has recently been determined that the cell death accompanying hypothermic and cryogenic storage involves an apoptotic component. U.S. Pat. No. 6,045,990, incorporated herein by reference, demonstrates, in part, that survival and recovery from cryopreservation can be enhanced by the inclusion of anti-apoptotic agents in the preservation medium.
There is a need in the art for improved methods of hypothermic and cryogenic preservation.